1. Field of the Invention
This invention relates generally to a control material useful in hemoglobin determination and, more specifically, this invention relates to a method for preparing a stable, nonturbid hemoglobin control material which comprises oxyhemoglobin with only traces of methemoglobin.
2. Description of the Prior Art
The determination of both total hemoglobin and glycosylated hemoglobin, specifically hemoglobin Alc (HbAlc), which is also referred to as glycosylated oxyhemoglobin, is recognized as clinically important in the diagnosis and monitoring of control in diabetic patients. The concentration of hemoglobin Alc is recognized as being directed related to time-averaged blood glucose levels and is correlated with various stages of metabolic control in diabetic patients.
Normal, nondiabetic patients have a HbAlc level of between about 4-8% of total hemoglobin, while the HbAlc level in diabetic patients may range up to about 20% of total hemoglobin. Hypoglycemic patients correspondingly have a HbAlc level below about 3% of total hemoglobin.
Various clinical methods have been developed to provide practical means for assessing the HbAlc level in a sample of a patient's blood. Such methods include both electrophoretic and chromatographic techniques.
In both electrophoretic and chromatographic methods, standards and control materials having known glycosylated hemoglobin concentrations are necessary in order to assure day-to-day consistency of test results and to provide a reference for calibration of equipment.
One prior chromatographic procedure for determining glycosylated hemoglobin levels is a microcolumn chromatography method embodied in a test kit sold by Isolab, Inc. of Akron, Ohio under the trademark "Quik-Sep.RTM. Fast Hemoglobin.TM." system. In the Isolab Quik-Sep.RTM. system, both total and glycosylated hemoglobin are determined. In this procedure, whole blood is hemolyzed and placed on a disposable column containing an ion exchange resin. A so-called "fast" hemoglobin fraction is eluted from the column with a first buffer solution, and the remaining ("other") hemoglobins are eluted with a second buffer solution. The "fast" hemoglobin consists of glycosylated hemoglobins. After dilution of the second fraction with water, the optical density of each eluate fraction is determined with a standard spectrophotometer, and compared with the optical density of a standard solution having a known amount of hemoglobin.
Another commercially available column chromatography test method for determining glycosylated hemoglobin is sold by BioRad Laboratories, Richmond, Calif. under the trademark "BioRad Hemoglobin Alc Column Test".
The accuracies of the microcolumn chromatography test procedures referred to above are subject to day-to-day variations due to changes in temperature, or changes in the pH of the ion exchange resin. Therefore, it is important that a reliable standard or control material be available to provide a reference for calibration of the column.
Prior hemoglobin control materials, some of which are provided in lyophilized form, have typically contained undesirably high amounts of cyanomethemoglobin, methemoglobin or cyanohemoglobin, the presence of which adversely affects the accuracy of glycosylated hemoglobin determination, since these forms of hemoglobin travel through an ion exchange column at different rates than does glycosylated hemoglobin, and their absorption spectra differ from that of "native" hemoglobin.
Furthermore, prior liquid or reconstituted dry control materials are generally turbid, contain particulate matter, and/or have low shelf lives. Turbidity is generally caused by the presence of lipids or cell stroma. Also, prior controls sometimes contain preservatives in order to enhance shelf life. Such preservatives are generally undesirable because they bind with or dissociate the hemoglobin molecule, or otherwise adversely affect hemoglobin present in the control material.
As a result, prior hemoglobin controls and standards often contain a modified form of hemoglobin, whose absorption characteristics are dissimilar to natural hemoglobin, or which are unstable due to autooxidation.